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Options to treat early-onset scoliosis include guided-growth systems with sliding action between rods and pedicle screws. The wear was previously measured in an in vitro test, and in this in vivo rabbit model, we evaluated the local and systemic biological response to the stainless steel debris. Compared to the previous study, a relatively higher volume of representative wear particles with a median particle size of 0.84 μm were generated. Bolus dosages were injected into the epidural space at L4-L5 for a minimum of 36 rabbits across three treatment groups (negative control, 1.5 mg, and 4.0 mg) and two timepoints (12 and 24 weeks). Gross pathology evaluated distant organs and the injection site with a dorsal laminectomy to examine the epidural space and dosing site. Peri-implanted particle tissues were stained for immunohistochemical and quantitatively analyzed for IL-6 and TNF-α cytokines. Based on ISO 10993-6:2007 scoring, particles in the high-dose group were primarily non-irritant (12 weeks) with one slightly irritant. At 24 weeks, inflammatory cell infiltration was non-existent to minimal with all groups considered non-irritant at the injection site. Material characterization confirmed that particles detected in distant organs were stainless steel or contaminants. At 12 weeks, stainless steel groups demonstrated statistically increased amounts of cytokine levels compared to control but there was a statistical decrease for both at 24 weeks. These findings indicate that stainless steel wear debris, comparable to the expected usage from a simulated growth guidance system, had no discernible untoward biological effects locally and systemically in an animal model. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1980-1990, 2018.

Introduction Lumbar disk herniation may result in a radiculopathic pattern of symptoms. Consideration for a primary biochemical inducement of pain over a mechanical mechanism is a contemporary topic of spinal research. However, the exact pathomechanism by which a degenerative intervertebral disk leads to neural inflammation and pain has not been determined. Using modern techniques of chemical analysis, biochemical markers can be identified which participate in the degenerative cascade, and possibly with the onset of pain. The purpose of this research is to identify potential biochemical markers through a novel technique of epidural space lavage that may be helpful in understanding the pathogeneses of pain in the presence of intervertebral disk degeneration and herniation. Methods Fifty consecutive patients with acute radiculopathy secondary to a symptomatic herniated lumbar intervertebral disk or spinal stenosis, and who were indicated for epidural steroid injection were identified. Additionally, 3 volunteers with no history of back pain or radiculopathy volunteered to undergo epidural lavage. After needle insertion, a lavage followed by fluid aspiration of the epidural space at the level of the disc herniation, in the case of the symptomatic patients, was performed using normal saline, before the instillation of corticosteroids. The fluid samples were frozen at -20 degrees C until analysis. A biochemical evaluation for a battery of cytokines was undertaken (IL-1beta, IL-1ra, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p40, IL-13, IL-15, IL-17, TNF-alpha, IFN-alpha, IFN-gamma, GM-CSF, MIP-1alpha, MIP-1beta, IP-10, MIG, Eotaxin, RANTES, and MCP-1, and neuropeptides) using high-resolution multiplex bead immunoassays and enzyme-linked immunosorbent assay (ELISA). Additionally, polyacrylamide gel electrophoresis was carried out to verify the presence of serum proteins. Results Despite the presence of amino acids/serum proteins in the epidural lavage fluid, none of the aforementioned mediators were isolated in a quantifiable concentration using the ELISA techniques with >5 pg/mL resolution. Discussion The current proteomics array technology was not able to detect critical levels of biochemical markers present in the epidural space through the mentioned lavage technique. This lack of detection could be due to the absence of the factors in this environment or the inability of the technique to obtain or detect factors which may be present. Conclusion Although a novel approach, the current study was unable to identify the presence of a series of inflammatory peptides in the epidural lavage of patients with symptomatic radicular pain due to herniated disc disease. We recommend alternative experimental designs than the one we pursued for definitively identifying potential sources of pain generators.

Total joint arthroplasty is a well accepted orthopaedic procedure for the treatment of many pathological joint conditions, but the release of metallic wear debris such as cobalt chromium (CoCr) microparticles can lead to pain, osteolysis, and implant failure. Our studies of periprosthetic tissue samples from patients undergoing revision surgery have shown that these particles induce an M2 macrophage phenotype as evidenced through gene expression analysis and CD68 immunostaining. We used an intra-articular murine knee model to further examine the synovial tissue response to CoCr and have shown that a single high dose (10mg) inoculation of CoCr microparticles triggers a robust type 2 immune response characterized by the presence of alternatively activated macrophages, neutrophils, and eosinophils 2 days after injection. We further examined the long term effects of repeated alternate day low dose (0.5mg) injection of CoCr microparticles on synovial tissues over 14 days. Tissues exposed to CoCr microparticles were found to have increased clinical scores for inflammation with loss of cartilage and bone tissue at day 14 when compared to controls. Together these studies indicate that exposure of synovial tissues to CoCr induces an innate type 2 immune response with long term hard tissue damage. These studies may ultimately lead to the identification of targeted therapeutic options to locally treat implant associated pain and potentially increase implant longevity.

Wear debris microparticle release associated with total joint arthroplasty may contribute to harmful inflammation, osteolysis and implant failure. We examined the role of different molecular signaling pathways in microparticle-mediated inflammation. Intraperitoneal inoculation of cobalt chrome (CoCr) in mice induced a robust innate type 2 response with increases in Th2 cytokines, M2 macrophages, eosinophils and neutrophils compared to controls given vehicle only. Furthermore, this type 2 innate particle-induced response was independent of MYD88/TRIF and partially dependent on Caspase-1 signaling pathways. Blockade of the PGE-2 signaling pathway by administration of Aspirin or Celecoxib prior to CoCr inoculation caused a significant reduction in both neutrophils and macrophages with no effect on eosinophils and Th2 cytokines compared to the mice treated with CoCr + vehicle. Blockade of the SYK signaling pathway with the inhibitor BAY61-3606 or the BTK signaling pathway with Ibrutinib completely abrogated the CoCr-mediated innate inflammation and Th2 cytokines. These studies indicate that the type 2 immune response caused by solid particles is partially modulated by Caspase-1 and PGE-2 pathways and largely abrogated by blockade of upstream SYK and BTK signaling pathways. These studies provide potential targets for controlling the wear debris-mediated inflammation in synovial tissues that can contribute to aseptic loosening and implant failure.

Orthopaedic wear debris may contribute to harmful inflammation and implant failure. In this study, we examined whether Cobalt Chrome (CoCr) can trigger inflammatory responses under aseptic conditions. Intra-peritoneal injection of mice with CoCr microparticles induced a type 2 innate immune response with marked increases in M2 macrophages, neutrophils and eosinophils in WT and MYD88/TRIF-/- mice. In contrast, although Th2 cytokine MRNA elevations were intact, a marked decrease in neutrophil and M2 macrophages occurred in Caspase-1-/- mice compared to wild type mice. SYK pathway blockade with the inhibitor BAY 61-3606 caused a marked general decrease in type 2 inflammation. Adjuvant properties of CoCr particles were assessed by immunizing mice with CoCr+OVA peptide after transfer of CFSE-labeled OVA specific DO11.10 CD4+ T cells. After 7 days, marked increases in OVA-specific T cell proliferation and IL-4 production were observed compared to mice immunized with OVA alone. To more closely approximate effects of wear debris in joint replacements, localized immune cell infiltrates were examined after intra-articular knee injections of CoCr particles. Infiltration of specific immune cells and mRNA expression were similar to peritoneal inoculation. These studies indicate that the type 2 immune response is triggered by solid particles, independent of toll like receptor signalling, and differentially modulated by Inflammasome and SYK signaling pathways

Wear debris particles associated with prosthetic devices may cause inflammation. In our previous study, we demonstrated that micrometer size Titanium particles induce a robust innate and adaptive type 2 in vivo immune response. In this study, we examined the size and mechanisms of particle induced type 2 inflammation, using Cobalt Chrome (COCR). Intra-peritoneal injection of 50 mg COCR (>10micron) still induced a type 2 innate response with marked increases in M2 macrophages, neutrophil and eosinophils independent of MYD88/TRIFF signaling pathways. We observed a marked decrease in the infiltration of neutrophil and M2 macrophages in Caspase-1-/- mice compared to wild type mice suggesting an essential role for inflammasome-mediated pathways. Intra peritoneal injection with smaller size COCR particles (10micron). We also examined the adjuvant nature of COCR particles by immunizing mice with COCR microparticles + OVA peptide after transfer of CFSE-labeled OVA specific DO11.10 CD4+ T cells. After 7 days marked increases in OVA-specific T cell proliferation and IL-4 production was observed compared to mice immunized with OVA alone. These studies indicate that induction of the type 2 immune response is triggered by solid particles alone similar in size to macroparasites, independently of MYD88 signaling and at least partially dependent on inflammasome activation.